Project 3. Cardiac Differentiation from Human Embryonic Stem Cells (Charles E. Murry, Project Leader) The long term goals of this work are to understand pathways controlling differentiation of cardiomyocytes from human embryonic stem cells (hESCs). These pluripotent cells offer a system for basic research in human development and cell biology, as well as potential applications for tissue repair and regeneration. Traditional ESC differentiation systems, such as embryoid bodies, are heterogeneous and yield cardiomyocytes at low efficiency, typically <1%. This lack of purity and low yield has significantly impeded research. In collaboration with colleagues at Geron, we recently developed a system for directed differentiation of hESCs, using Activin A and BMP4, which yields -50% cardiomyocytes before purification and >95% myocytes after purification. Furthermore, in collaboration with Dr. Moon's laboratory (Project 2), we have shown a potent role for Wnt/b-catenin signaling in control of cardiogenesis in mouse ESCs. The ability to efficiently control ESC differentiation makes possible systematic studies into the mechanism of human cardiac differentiation and could yield populations of cells, such as pre-cardiac mesodermal progenitors, that have remained elusive due to technical limitations. The current proposal therefore focuses on optimizing these regimens and understanding key molecular and cellular steps in cardiac differentiation from hESCs.